The invention relates to a method for isolating a target biological material contained in a sample, using a capture phase comprising an organic molecule containing at least one reactive function, and at least one protein material capable of recognizing or binding, specifically and directly or indirectly, to the target biological material, said protein material containing a specific site for covalently binding to the reactive function of the organic molecule.
A method for immobilizing a protein on a solid support, by means of reactive functions, in order to stimulate the proliferation or growth of T cells and the production of killer lymphocytes is known from document EP-A-0,319,012. To this end, the protein has, at its C-terminal end, a covalent-binding site for binding to said reactive functions, which consists of an amino acid sequence in which are distributed two to four lysine residues.
The problem posed by the covalent-binding site of this prior art lies in the fact that, in a diagnostic application, the results of the isolation of a target biological material with which it is capable of binding are similar to those obtained with a protein material which does not contain such a binding site.
According to the invention, a method is provided for isolating a target biological material by using at least one capture phase which comprises a protein material containing a covalent-binding site which allows efficient orientation of said protein material, and leads to sensitive, high-quality detection of said biological material.
Thus, the method of the invention, for isolating a target biological material contained in a sample, comprises the following steps:
a capture phase is provided, which comprises an organic molecule containing at least one reactive function and at least one protein material capable of recognizing or binding, specifically and directly or indirectly, to the target biological material, said protein material containing a specific site for covalent binding to the reactive function of the organic molecule, which consists of at least one tag comprising at least six contiguous lysine or lysine-based residues,
said target biological material is placed in contact with at least one capture phase, and
the target biological material bound to the capture phase is detected.
According to the invention, the expression xe2x80x9cisolating a biological materialxe2x80x9d means the binding, separation, isolation, detection and/or quantification of this material, the enrichment of a fraction with target biological material, according to a qualitative and/or quantitative, specific or aspecific binding method.
According to one variant of the method, the capture phase can also comprise a label, and, in this case in particular, it can consist of a detection phase.
According to another variant of the method, a detection phase is also provided, which comprises an organic molecule containing at least one reactive function, at least one protein material capable of recognizing or binding, specifically and directly or indirectly, to said target biological material, and a label, said protein material containing a specific site for covalent binding to the reactive function of the organic molecule, which consists of at least one tag comprising at least six contiguous lysine or lysine-based residues.
In this case, the organic molecules in the capture phase and in the detection phase, respectively, can be identical or different, and the protein materials in the capture phase and in the detection phase, respectively, can be identical or different.
As is understood by the invention, a sample comprises any sample capable of containing a biological material, in particular a sample such as that obtained fromxe2x80x94a biological fluid, a sample of food origin, or a cell culture.
The sample consists of all or part of another sample: in particular it can consist of an aliquot or a dilution.
A protein material according to the invention comprises proteins, in particular recombinant proteins, especially antigens, antibodies and peptides such as synthetic peptides. The method of the invention may also be carried out with a material such as peptide analogues of nucleic acids (PNA).
The term xe2x80x9corganic moleculexe2x80x9d means a molecule of variable size; thus, this refers equally to a small molecule such as a hapten and to a macromolecule such as a polymer.
As examples of haptens, mention may be made of a hormone, a vitamin, such as biotin, or a medicinal product. In this case, the method of the invention can comprise, before the step of detecting the target biological material, a step of binding the organic molecule to a carrier molecule. Preferably, the hapten is biotin and the carrier molecule is avidin.
A polymer as used according to the invention is a polymer in particulate or in linear form. It can be a homopolymer chosen in particular from polylysine and polytyrosine, or a copolymer chosen in particular from maleic anhydride copolymers, N-vinylpyrrolidone copolymers, natural or synthetic polysaccharides, polynucleotides and amino acid copolymers such as enzymes. Advantageously, it is a copolymer chosen from maleic anhydride/methyl vinyl ether copolymer, N-vinylpyrrolidone/N-acryloxysuccinimide copolymer, poly-6-aminoglucose, and enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase or derivatives thereof bearing at least one reactive function.
The expression xe2x80x9creactive function of the organic moleculexe2x80x9d means either a reactive function chosen in particular from ester, halocarbonyl, sulfhydryl, disulfide, epoxide, haloalkyl and aldehyde functions; or a function which can be activated by an activating agent such as carbodiimides or homo- or heterobifunctional compounds. By way of example, an activatable function is chosen in particular from acid, amine and hydroxyl functions.
The covalent-binding sites defined above can exist naturally in the protein material. Alternatively, they can be xe2x80x9cincorporatedxe2x80x9d beforehand into the protein material, in the form of a tag, according to techniques which are well known to those skilled in the art, such as the technique used to purify proteins by the IMAC (immobilized metal ion-affinity chromatography) method on resins (1, 2). By way of example, such sites can be incorporated into a protein material, and in particular a protein, by genetic engineering in order to obtain recombinant proteins.
A tag can be defined as an amino acid sequence which is incorporated into, i.e. added to, the original structure of the protein material, which is introduced into a preferred place in said original structure in order to allow it to be exposed in a relevant manner, in particular with regard to its covalent binding to the organic molecule.
In accordance with the invention, a covalent-binding site of the protein material considered can consist of a tag comprising six or more lysine or lysine-based residues, and optionally other amino acids, or can consist of several of said tags.
The term xe2x80x9clysine-basedxe2x80x9d means that the lysine can be chemically modified, provided that these modifications essentially preserve or even enhance the specificity of the covalent-binding site. Examples which may be mentioned are the replacement of L-lysine with D-lysine, and vice-versa; a modification of the lysine side chain, such as an acetylation of the amine function or an esterification of the carboxyl function; a modification of the peptide bonds such as, for example, carba, retro, inverso, retro-inverso, reduced and methylenoxy bonds.
The tag(s) described above can be found in any place in the primary structure of the protein material. Preferably, it is located at the N- or C-terminal end of the protein material.
According to the present invention, various methodes are also defined for isolating a target biological material contained in a sample, depending, in particular, on its nature:
if the target biological material is an antibody, the protein material comprises an antigen which specifically recognizes said antigen;
if the target biological material is an antigen, the protein material comprises an antibody which specifically recognizes said antigen.
Other subjects of the invention are outlined below.
Thus, the invention relates to a phase for capturing a target biological material which comprises an organic molecule containing at least one reactive function and at least one protein material capable of recognizing or binding, specifically and directly or indirectly, to the target biological material, said protein material containing a specific site for covalent binding to the reactive function of the organic molecule, which consists of at least one tag comprising at least six contiguous lysine residues; preferably, the organic molecule is a polymer or a hapten.
An advantageous polymer is particulate or linear and chosen in particular from homopolymers such as polylysine or polytyrosine; and copolymers such as maleic anhydride copolymers, N-vinylpyrrolidone copolymers, natural or synthetic polysaccharides, polynucleotides and amino acid copolymers such as enzymes. Even more advantageously, the polymer is chosen from maleic anhydride/methyl vinyl ether copolymer, N-vinylpyrrolidone/N-acryloxysuccinimide copolymer, poly-6-aminoglucose, horseradish peroxidase (HRP) and alkaline phosphatase.
If the organic molecule is a hapten, the capture phase can also comprise a suitable carrier molecule. Preferably, the hapten is biotin and, if necessary, the carrier molecule is avidin.
Advantageously, the tag as defined above is placed at the N- or C-terminal end of the protein material.
The reactive function of the organic molecule is chosen in particular from ester, acid, halocarbonyl, sulfhydryl, disulfide, epoxide, haloalkyl and aldehyde functions.
The invention also relates to a phase for detecting a target biological material, having the same characteristics as the above capture phase and also comprising a label.
As indicated above, the organic molecule can be a polymer or a hapten. According to the second possibility, the organic molecule consisting of the hapten also represents the label.
The label for the detection phase is preferably chosen from the group consisting of an enzyme, a protein, a peptide, an antibody, a hapten such as biotin or iminobiotin, a fluorescent compound such as rhodamine, a radioactive compound, a chemiluminescent compound, an electron-density component, a magnetic component and the like.
Another subject of the invention is a reagent for isolating a target biological material, comprising a capture phase of the invention and/or a detection phase of the invention.
Advantageously, the capture phase is bound, directly or indirectly, to a solid support, by passive absorption or by covalency.
The solid support can be in any suitable form, such as a plate, a cone, a bead which is optionally radioactive and/or fluorescent and/or magnetic and/or conductive, a bar, a tube of glass, a well, a sheet, a chip or the like. It is chosen from among polystyrenes, styrene/butadiene copolymers, styrene/butadiene copolymers mixed with polystyrenes, polypropylenes, polycarbonates, polystyrene/acrylonitrile copolymers, styrene/methyl methylmethacrylate copolymers, from among synthetic and natural fibres, and from among polysaccharides and cellulose derivatives, glass, silicon and derivatives thereof.
As will be demonstrated in the experimental section below, the Authors have obtained monoclonal antibodies directed against the tags as defined above and in particular against the tag which consists of a sequence of six lysine residues.
It is thus possible to envisage the use of such a tag in a method for isolating a biological material, according to which:
a capture phase is provided, said target biological material is placed in contact with at least the capture phase, and the target biological material bound to the capture phase is detected, according to which method the capture phase comprises an organic molecule which comprises or consists of an anti-tag antibody and at least one protein material capable of recognizing or of binding, specifically and directly or indirectly, to the target biological material, said protein material containing a specific site for binding to the organic molecule, which consists of at least one tag comprising at least six contiguous lysine or lysine-based residues.
Such a method can also comprise the use of a detection phase, which has the characteristics of the capture phase as has just been described, and also comprising a label. This label can advantageously consist of a said antibody.